Instability of ackA (Acetate Kinase) Mutations and Their Effects on Acetyl Phosphate and ATP Amounts in Streptococcus pneumoniae D39

Abstract

ABSTRACT Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA , respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H 2 O 2 ) by pyruvate oxidase, which is encoded by spxB . To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB , pta , and ackA mutations and determined their effects on ATP, AcP, and H 2 O 2 production. Unexpectedly, Δ ackA mutants were unstable and readily accumulated primary suppressor mutations in s pxB or its positive regulator, spxR , thereby reducing H 2 O 2 and AcP levels, and secondary capsule mutations in cps2E or cps2C . Δ ackA Δ spxB mutants contained half the cellular amount of ATP as a Δ spxB or spxB + strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that Δ ackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H 2 O 2 may require energy. Starting with a Δ spxB mutant, we constructed Δ pta , Δ ackA , and Δ pta Δ ackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK Spn , CiaRH Spn , and LiaSR Spn TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.